MEC1P IS ESSENTIAL FOR PHOSPHORYLATION OF THE YEAST DNA-DAMAGE CHECKPOINT PROTEIN DDC1P, WHICH PHYSICALLY INTERACTS WITH MEC3P

Checkpoints prevent DNA replication or nuclear division when chromosom es are damaged. The Saccharomyces cerevisiae DDC1 gene belongs to the RAD17, MEC3 and RAD24 epistasis group which, together with RAD9, is pr oposed to act at the beginning of the DNA damage checkpoint pathway. D dc1p is periodically phosphorylated during unperturbed cell cycle and hyperphosphorylated in response to DNA damage. We demonstrate that Ddc 1p interacts physically in vivo with Mec3p, and this interaction requi res Rad17p, We also show that phosphorylation of Ddc1p depends on the key checkpoint protein Mec1p and also on Rad24p, Rad17p and Mec3p. Thi s suggests that Mec1p might act together with the Rad24 group of prote ins at an early step of the DNA damage checkpoint response. On the oth er hand, Ddc1p phosphorylation is independent of Rad53p and Rad9p, Mor eover, while Ddc1p is required for Rad53p phosphorylation, it does not play any major role in the phosphorylation of the anaphase inhibitor Pds1p, which requires RAD9 and MEC1, We suggest that Rad9p and Ddc1p m ight function in separated branches of the DNA damage checkpoint pathw ay, playing different roles in determining Mec1p activity and/or subst rate specificity.